Guy P. Richardson

Stereocilia are rod-like mechanosensory projections consisting of unidirectionally oriented actin filaments that extend into the inner ear hair cell cytoskeleton, forming dense rootlets. Taperin (TPRN) localizes to the narrowed-down base of stereocilia, where they pivot in response to sound and gravity. We show that TPRN-deficient mice have progressive deafness characterized by gradual asynchronous retraction and fusion of outer and inner hair cell stereocilia, followed by synaptic abnormalities. Stereocilia that lack TPRN develop warped rootlets with gradual loss of TRIOBP-5 and ANKRD24 from mechanosensory rows starting postnatally. In contrast, TPRN overexpression causes excessive F-actin bundling, extra rows, and over-elongation of stereocilia during development. Purified full-length mouse TPRN cross-links F-actin into bendable bundles reflecting in vivo data. This F-actin–bundling ability is attributed to the TPRN N-terminal region. TPRN interacts with the membrane receptor PTPRQ, connecting the F-actin core to the plasma membrane, stabilizing stereocilia. Thus, TPRN is a specialized F-actin bundler strategically located to augment stereocilia rootlet formation and their pivot point flexibility for sustained sound-induced deflections.

The styryl dye FM1-43 is widely used to study endocytosis but behaves as a permeant blocker of the mechano-electrical transducer (MET) channel in sensory hair cells, loading rapidly and specifically into the cytoplasm of hair cells in a MET channel-dependent manner. Patch clamp recordings of mouse outer hair cells (OHCs) were used to determine how a series of structural modifications of FM1-43 affect MET channel block. Fluorescence microscopy was used to assess how the modifications influence hair-cell loading in mouse cochlear cultures and zebrafish neuromasts. Cochlear cultures were also used to evaluate otoprotective potential of the modified FM1-43 derivatives. Structure-activity relationships reveal that the lipophilic tail and the cationic head group of FM1-43 are both required for MET channel block in mouse cochlear OHCs; neither moiety alone is sufficient. The extent of MET channel block is augmented by increasing the lipophilicity/bulkiness of the tail, by reducing the number of positive charges in the head group from two to one, or by increasing the distance between the two charged head groups. Loading assays with zebrafish neuromasts and mouse cochlear cultures are broadly in accordance with these observations but reveal a loss of hair-cell specific labelling with increasing lipophilicity. Although FM1-43 and many of its derivatives are generally cytotoxic when tested on cochlear cultures in the presence of an equimolar concentration of the ototoxic antibiotic gentamicin (5 µM), at a 10-fold lower concentration (0.5 µM), two of the derivatives protect OHCs from cell death caused by 48 h-exposure to 5 µM gentamicin.

Purpose A probe that binds to unfixed collagen fibrils was used to image the shapes and fibrous properties of the TM and BM. The probe (CNA35) is derived from the bacterial adhesion protein CNA. We present confocal images of hydrated gerbil TM, BM, and other cochlear structures stained with fluorescently labeled CNA35. A primary purpose of this article is to describe the use of the CNA35 collagen probe in the cochlea. Methods Recombinant poly-histidine-tagged CNA35 was expressed in Escherichia coli, purified by cobalt-affinity chromatography, fluorescence labeled, and further purified by gel filtration chromatography. Cochleae from freshly harvested gerbil bullae were irrigated with and then incubated in CNA35 for periods ranging from 2 h — overnight. The cochleae were fixed, decalcified, and dissected. Isolated cochlear turns were imaged by confocal microscopy. Results The CNA35 probe stained the BM and TM, and volumetric imaging revealed the shape of these structures and the collagen fibrils within them. The limbal zone of the TM stained intensely. In samples from the cochlear base, intense staining was detected on the side of the TM that faces hair cells. In the BM pectinate zone, staining was intense at the upper and lower boundaries. The BM arcuate zone was characterized by a prominent longitudinal collagenous structure. The spiral ligament, limbus and lamina stained for collagen, and within the spiral limbus the habenula perforata were outlined with intense staining. Conclusion The CNA35 probe provides a unique and useful view of collagenous structures in the cochlea.

Sensory‐independent Ca ²⁺ spiking regulates the development of mammalian sensory systems. In the immature cochlea, inner hair cells (IHCs) fire spontaneous Ca ²⁺ action potentials (APs) that are generated either intrinsically or by intercellular Ca ²⁺ waves in the nonsensory cells. The extent to which either or both of these Ca ²⁺ signalling mechansims are required for IHC maturation is unknown. We find that intrinsic Ca ²⁺ APs in IHCs, but not those elicited by Ca ²⁺ waves, regulate the maturation and maintenance of the stereociliary hair bundles. Using a mouse model in which the potassium channel Kir2.1 is reversibly overexpressed in IHCs ( Kir2.1‐ OE), we find that IHC membrane hyperpolarization prevents IHCs from generating intrinsic Ca ²⁺ APs but not APs induced by Ca ²⁺ waves. Absence of intrinsic Ca ²⁺ APs leads to the loss of mechanoelectrical transduction in IHCs prior to hearing onset due to progressive loss or fusion of stereocilia. RNA‐sequencing data show that pathways involved in morphogenesis, actin filament‐based processes, and Rho‐GTPase signaling are upregulated in Kir2.1‐ OE mice. By manipulating in vivo expression of Kir2.1 channels, we identify a “critical time period” during which intrinsic Ca ²⁺ APs in IHCs regulate hair‐bundle function.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC’s mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin–Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.

Key points: The aim was to determine whether detachment of the tectorial membrane (TM) from the organ of Corti in Tecta/Tectb^−/− mice affects the biophysical properties of cochlear outer hair cells (OHCs). Tecta/Tectb^−/− mice have highly elevated hearing thresholds, but OHCs mature normally. Mechanoelectrical transducer (MET) channel resting open probability (P_o) in mature OHC is ∼50% in endolymphatic [Ca²⁺], resulting in a large standing depolarizing MET current that would allow OHCs to act optimally as electromotile cochlear amplifiers. MET channel resting P_o in vivo is also high in Tecta/Tectb^−/− mice, indicating that the TM is unlikely to statically bias the hair bundles of OHCs. Distortion product otoacoustic emissions (DPOAEs), a readout of active, MET‐dependent, non‐linear cochlear amplification in OHCs, fail to exhibit long‐lasting adaptation to repetitive stimulation in Tecta/Tectb^−/− mice. We conclude that during prolonged, sound‐induced stimulation of the cochlea the TM may determine the extracellular Ca²⁺ concentration near the OHC's MET channels. The tectorial membrane (TM) is an acellular structure of the cochlea that is attached to the stereociliary bundles of the outer hair cells (OHCs), electromotile cells that amplify motion of the cochlear partition and sharpen its frequency selectivity. Although the TM is essential for hearing, its role is still not fully understood. In Tecta/Tectb^−/− double knockout mice, in which the TM is not coupled to the OHC stereocilia, hearing sensitivity is considerably reduced compared with that of wild‐type animals. In vivo, the OHC receptor potentials, assessed using cochlear microphonics, are symmetrical in both wild‐type and Tecta/Tectb^−/− mice, indicating that the TM does not bias the hair bundle resting position. The functional maturation of hair cells is also unaffected in Tecta/Tectb^−/− mice, and the resting open probability of the mechanoelectrical transducer (MET) channel reaches values of ∼50% when the hair bundles of mature OHCs are bathed in an endolymphatic‐like Ca²⁺ concentration (40 μM) in vitro. The resultant large MET current depolarizes OHCs to near –40 mV, a value that would allow optimal activation of the motor protein prestin and normal cochlear amplification. Although the set point of the OHC receptor potential transfer function in vivo may therefore be determined primarily by endolymphatic Ca²⁺ concentration, repetitive acoustic stimulation fails to produce adaptation of MET‐dependent otoacoustic emissions in vivo in the Tecta/Tectb^−/− mice. Therefore, the TM is likely to contribute to the regulation of Ca²⁺ levels around the stereocilia, and thus adaptation of the OHC MET channel during prolonged sound stimulation.

The aging cochlea is subjected to a number of pathological changes to play a role in the onset of age‐related hearing loss (ARHL). Although ARHL has often been thought of as the result of the loss of hair cells, it is in fact a disorder with a complex etiology, arising from the changes to both the organ of Corti and its supporting structures. In this study, we examine two aging pathologies that have not been studied in detail despite their apparent prevalence; the fusion, elongation, and engulfment of cochlear inner hair cell stereocilia, and the changes that occur to the tectorial membrane (TM), a structure overlying the organ of Corti that modulates its physical properties in response to sound. Our work demonstrates that similar pathological changes occur in these two structures in the aging cochleae of both mice and humans, examines the ultrastructural changes that underlie stereocilial fusion, and identifies the lost TM components that lead to changes in membrane structure. We place these changes into the context of the wider pathology of the aging cochlea, and identify how they may be important in particular for understanding the more subtle hearing pathologies that precede auditory threshold loss in ARHL.

CEACAM16 is a non-collagenous protein of the tectorial membrane, an extracellular structure of the cochlea essential for normal hearing. Dominant and recessive mutations in CEACAM16 have been reported to cause postlingual and progressive forms of deafness in humans. In a previous study of young Ceacam16^βgal/βgal null mutant mice on a C57Bl/6J background, the incidence of spontaneous otoacoustic emissions (SOAEs) was greatly increased relative to Ceacam16^+/+ and Ceacam16^+/βgal mice, but auditory brain-stem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were near normal, indicating auditory thresholds were not significantly affected. To determine if the loss of CEACAM16 leads to hearing loss at later ages in this mouse line, cochlear structure and auditory function were examined in Ceacam16^+/+, Ceacam16^+/βgal and Ceacam16^βgal/βgal mice at 6 and 12 months of age and compared to that previously described at 1 month. Analysis of older Ceacam16^βgal/βgal mice reveals a progressive loss of matrix from the core of the tectorial membrane that is more extensive in the apical, low-frequency regions of the cochlea. In Ceacam16^βgal/βgal mice at 6–7 months, the DPOAE magnitude at 2f1-f2 and the incidence of SOAEs both decrease relative to young animals. By ∼12 months, SOAEs and DPOAEs are not detected in Ceacam16^βgal/βgal mice and ABR thresholds are increased by up to ∼40 dB across frequency, despite a complement of hair cells similar to that present in Ceacam16^+/+ mice. Although SOAE incidence decreases with age in Ceacam16^βgal/βgal mice, it increases in aging heterozygous Ceacam16^+/βgal mice and is accompanied by a reduction in the accumulation of CEACAM16 in the tectorial membrane relative to controls. An apically-biased loss of matrix from the core of the tectorial membrane, similar to that observed in young Ceacam16^βgal/βgal mice, is also seen in Ceacam16^+/+ and Ceacam16^+/βgal mice, and other strains of wild-type mice, but at much later ages. The loss of Ceacam16 therefore accelerates age-related degeneration of the tectorial membrane leading, as in humans with mutations in CEACAM16, to a late-onset progressive form of hearing loss.